immunohematology and blood banking techniques class notes
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Class notesTRANSCRIPT
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IMMUNOHEMATOLOGY &
BLOOD BANKING TECHNIQUES
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INTRODUCTION:
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IMMUNO HEMATO LOGY
IMMUNOHEMATOLOGY
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DEFINITIONS:
Protection against infection or disease caused by foreign
particles.
IMMUNITY:
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4 Study of the cellular
components of the blood. HEMATOLOGY:
Study of the immune system and the immune response.
Study of antigen-antibody reactions in vivo.
IMMUNOLOGY:
Study of antigen-antibody reactions in vitro.
SEROLOGY:
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The Immune System
Three functions:
Defense
Homeostasis
Surveillance
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The Immune System
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Markers of Self
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Markers of Non-Self
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Markers of Self:
Major Histocompatibility Complex
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Organs of the Immune System
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Components:
Cells:
Lymphocytes: T-cells, B-cells & NK cells.
Phagocytic cells: N, E, B, Monocytes,
Macrophages, Dendritic cell, etc.
Chemical mediators:
Complement system.
Chemokines.
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Cells of the Immune System
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B Cells
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Antibody
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Immunoglobulins
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Antibody Genes
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T Cells
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Cytokines
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Killer Cells: Cytotoxic Ts and NKs
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Phagocytes and Their Relatives
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Phagocytes in the Body
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Complement
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Immunity: Active and Passive
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DEFINITIONS:
IMMUNOHEMATOLOGY:
DETECTION,
IDENTIFICATION, and/or
QUANTITATION of antibodies involved primarily with
red cells [although white cells and platelets may also
be involved].
Basically this branch of science related with red cell
antigens and their corresponding antibodies.
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IMMUNOHEMATOLOGY FACILITIES:
TRANSFUSION SERVICE:
Work primarily with patient's blood.
Primary areas of responsibility:
Blood typing. Antibody detection and
identification.
Compatibility testing (crossmatching).
Blood component therapy (hemotherapy).
Transfusion reaction workups.
Autoimmune hemolytic anemia workups.
Hemolytic disease of the newborn (HDN) workups.
Determining Rh immune globulin eligibility.
BLOOD BANK:
Works primarily with donor blood.
Major areas of responsibility:
Donor recruitment. Donor screening. Blood collection. Testing (typing,
infectious disease
screening).
Blood component preparation.
Component preservation. May provide reference
lab services.
May store rare donor blood.
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HISTORICAL OVERVIEW
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1616: Sir William Harvey Described circulation of blood.
1665: Richard Lower, English Physiologist 1st animal-to-
animal [dog to dog] blood transfusion.
1667: Jean Bapiste Denys Unsuccessful transfusion of
animal-to-human [sheep to human] blood transfusion.
1667 1818: Transfusions prohibited.
1818: James Blundell of England 1st successful human-to-
human blood transfusion. This species specific transfusion
had 50% success rate, the rest resulted in death.
1900: Karl Landsteiner Discovered the ABO blood
groups.
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Karl Landsteiner:
Described the ABO
Blood Groups.
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Karl Landsteiners discovery:
Discovered that the incompatibility of many
transfusion was due to certain factors on red cells
now known as Antigens.
Postulated two things:
Each species has unique species specific factors on
red dells.
Even in each species has some common and some
uncommon factors to each other.
Introduced the Immunological era of blood
transfusion began the era of scientific based
transfusion therapy foundation of
IMMUNOHEMATOLOGY as a science.
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1927: Landsteiner and Levine discovered M,N and P
system.
1939,40: Levine, Stetson, Landsteiner and Weiner
discovers Rh system and its role in erythroblastosis
foetalis (HDN).
1946-Kell system discovered by Coombs, Mourant and
Race.
1950-51: Duffy, Kidd, Lutheran system discovered.
Landsteiner and Alexanders lead to the discovery of
>800 Blood group systems.
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ANTIGENS:
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ANTIGEN is a substance that either
combines with an antibody or
is processed and binds to a T lymphocyte to
stimulate an IMMUNE RESPONSE.
IMMUNOGEN - An antigen that stimulates an immune
response.
HAPTENS - small chemical substances that must be
bound to a larger molecule to provide sufficient
molecular weight for stimulation of antibody
production.
Proteins Complex carbohydrates
Lipopolysaccharides.
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Properties of Molecules that Contribute to Immunogenicity
PROPERTY DESCRIPTION
Foreignness Non-self more likely to stimulate antibody production
Size >10,000 M.W.
Chemical composition
Proteinbest immune response. Complex carbohydratesecond best immune response. Lipids, Nucleic acid weak immune response.
Complexity More complex molecules produce better immune response.
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Antigen location:
Some antigens protrude from the cell surface, while
others are an integral part of the membrane.
Physical location impacts antibody stimulation as well
as the physical ability of the antigen to react with an
antibody once it is produced.
Red Blood Cell Antigens:
Red blood cell antigens and corresponding antibodies
provide the foundation for blood bank testing.
More than 20 blood group systems that contain
greater than 200 red blood cell antigens.
ABO and Rh antigens are matched between donor and
recipient.
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ANTIBODIES
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Protein.
Produced in response to stimulation with an antigen.
Specific for the stimulating antigen and will react with
that antigen.
IMMUNOGLOBULIN 5 classes, IgG, IgM, IgA, IgD &
IgE.
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Immunological principles:
Primary immunological components are antigens and
antibodies.
Cardinal rule for antigens and antibodies [blood
bank]: Antigens on RBC surfaces & Antibodies in
serum / plasma.
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Primary & Secondary Immune responses:
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Antigen-Antibody Reactions: Factors influencing: Each antibody reacts with the antigen that stimulated its
production. Specificity:
Noncovalent bonds. Bonding:
The fit of the antigen and antibody depend on compatible shapes that allow the antigen and antibody to physically
touch - a lock and key mechanism.
Physical fit:
Antigens and antibodies must be present in optimal concentrations; excess antibodies will result in a situation
known as prozone phenomenon.
Concentration of
antigen and
antibody:
Optimal temperature of reactivity for a specific antibody will expedite the combination of antigen and antibody.
Temperature:
Incubation time must be that which is optimal for the specific antibody. General guidelines are a range of 1560 minutes for optimal antigen-antibody attachment.
Time:
A pH range of 7.27.4 is maintained for most antigen-antibody reactions.
pH:
A net negative charge known as zeta potential surrounds the red cells. The reduction of this charge influences the ability
of antigen and antibody to combine.
Surface charge:
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LOCK & KEY CONCENTRATION
AGGLUTINATION
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Visualization of Ag-Ab reaction in BB:
2 methods:
Agglutination.
Hemolysis.
Precipitation.
Agglutination involves a particulate antigen or an antigen that
is attached to a particle (such as a red blood cell).
Agglutination occurs in when:
1. An antibody molecule attaches to a single antigen on a
single cell with one antigen-binding site.
2. The free arm of the immunoglobulin molecule attaches
to an antigen on a second red cell. This creates a
crosslink.
3. Multiple cross links create a lattice.
4. The lattice is visualized as agglutination.
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Grading of Agglutination:
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BLOOD GROUP GENETICS
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Chromosomes & Genes:
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Chromosomes & Genes:
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Chromosomes & Genes:
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Basic Principles:
Genetics: Study of Inheritance.
Inheritance of transmissible characteristics or
traits: such as blood group antigens found on red
blood cells.
Each parent contributes 1/2 of the genetic
information.
The genetic information is contained
on chromosomes composed of DNA
Humans have 23 pairs of chromosomes
a. 22 matched (autosomal) chromosomes and
b. 1pair of sex chromosomes.
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System Common Genes Located on
Chromosome
ABO A, B, O 9
Rh D, C, E, c, e 1
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Basic Principles:
Genes are the units of inheritance within the
chromosomes.
Alleles: At each loci, different forms of the genes. E.g.
ABO Blood Group System - A1, A
2, B, and O as common
alleles.
Genotype: Genetic composition for a particular trait.
Homozygous: When the two inherited alleles are
the same. E.g. OO / AA / BB.
Heterozygous: when the two inherited alleles are
different. E.g. AO / AB / BO.
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Basic Principles:
Phenotype: The expression of a genotype.
Dominant: The dominant gene will express itself if
present both in homozygous as well as
heterozygous state. E.g. Rh (D).
Co-dominant: Both the alleles express. E.g. A & B.
Recessive: They express in homozygous state only.
Amorph: No gene product. e.g. O.
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Basic Principles:
Dosage: In some blood group systems, persons
homozygous for an allele have MORE antigen on their
red cells than persons heterozygous for an allele.
Variation in antigen expression due to the number of
alleles present is called DOSAGE.
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BLOOD GROUP SYSTEMS
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HISTORICAL PERSPECTIVE:
In 1901, Karl Landsteiner used his blood and the
blood of his colleagues:
Mixed the serum of some individuals with cells of
others.
Discovered three groups A, B & O.
His colleagues discovered the 4th
group AB.
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LANDSTEINERS LAW / RULE:
ABO antigens on red cells and the reciprocal
agglutinating antibodies in the serum of the same
individual (e.g. A antigens on red blood cells and
anti-B in the serum).
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ABO & H SYSTEM ANTIGENS:
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ABO ANTIGENS:
Present on RBC surface.
Also on lymphocytes, thrombocytes, organs,
endothelial cells, and epithelial cells.
Detectable at 5 to 6 weeks of gestation.
Newborns - weaker antigens.
Fully developed by two to four years of age.
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ABO ANTIGENS:
One factor contributing to the difference in ABO
antigen strength between newborns and adults is the
number of branched oligosaccharides.
Adults - greater numbers of branched chains
compared to newborns - more linear chains.
The branched chains permit attachment of more
molecules to determine antigen specificity.
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Phenotype Number of Ag sites
A1 adult 8,10,000 to 1,170,000
A1 cord 2,50,000 to 3,70,000
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INHERITANCE:
Simple Mendelian fashion from an individuals
parents.
Each individual possesses a pair of genes.
FOUR genes H, A, B & O.
Hh Chromosome 19 HH / Hh / hh.
hh Bombay phenotype Oh.
A, B & O - Chromosome 9.
A and B genes produce a detectable products while
the O gene is an amorph.
The expression of the A and B genes is codominant.
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INHERITANCE:
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Gene Combination Phenotype
AO A
AA A
BO B
BB B
AB AB
OO O
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GENE PRODUCTS & BIOCHEMICAL COMPOSITION OF BLOOD GROUP SUBSTANCES:
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58 Basic common core structure - an oligosaccharide chain attached
to either a protein or a lipid molecule present on cell surface.
GENE TRANSFERASE
H -L-fucosyltransferase
A -3-N-acetyl-D-galactosaminyl
Transferase
B -3-D-acetyl-D-galactosyl
Transferase
O No Transferase.
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GENE PRODUCTS & BIOCHEMICAL COMPOSITION OF BLOOD GROUP SUBSTANCES:
The L-fucose is the immunodominant sugar for the H
antigen.
The H antigen serves as a precursor for A and B
antigens.
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SECRETOR STATUS
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Ability to secrete ABH antigens in body secretions.
Chromosome 19: Se / se [amorph]. Gene product L- fucosyltransferase. A & B transferases are found in the
secretions of A / B persons regardless
of their secretor status.
Secretors:
Nonsecretors:
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Saliva, Sweat, Tears, Semen, Serum & Amniotic fluid.
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SUBGROUPS OF A & B
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Subgroups of A:
2 major subgroups:
~ 80% A1
~ 20% A2.
2 major subgroups of AB:
~ 80% A1B
~ 20% A2B.
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Group A1 Group A2
Qualitative differences:
Reaction with Anti-A in Forward
Grouping
4+ 4+
Number of Antigen Sites-Adults 10,00,000 2,50,000
Number of Antigen Sites-
Newborn 3,00,000 1,40,000
Quantitative differences:
Reaction with Anti-A1 Positive Negative
Anti-A1 in serum Absent ? Present
-3-N-acetyl-D-galactosaminyl
Transferase Activity Normal Diminished
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A1 more antigens on the cell surface more
branched chain oligosaccharides than A2.
2 mutations Pro156Leu / Single nucleotide deletion
1060 reduced enzyme activity of A2.
Routine antisera NO DIFFERENCE in reaction.
The LECTIN Dolichos biflorus is used to obtain an
extract with anti-A1 specificity.
A2 individuals can develop antibodies to the A1
antigen.
Additional A subgroups: Aintr
, A3, A
x, A
m, A
end, A
el,and
Abantu
Fewer antigenic sites on their surface.
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Subgroups of B:
Subgroups of B are very rare and encountered less
frequently than subgroups of A.
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ABO ANTIBODIES:
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Antibodies directed against ABO antigens are the
most important antibodies in transfusion medicine.
It is the only example of a blood group where each
individual produces antibodies to antigens not
present on the red cells.
Newborns have NO ABO ANTIBODIES.
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REVERSE GROUPING of Cord blood / Newborn serum
indicates BLOOD GROUP OF THE MOTHER.
The child will begin antibody production and have a
detectable titre at 3 6 months of age peaks at 5
10 years of age.
Originally thought to be NATURAL ANTIBODIES
formed with no antigenic stimulus.
Proposed mechanism some naturally occurring
substances resemble A & B antigens and stimulate
production of complementary antibodies.
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Conditions with decreased levels of ABO antibodies:
Newborns and young infants Elderly individuals
Age related:
Congenital conditions Congenital hypogammaglobulinemia Congenital agammaglobulinemia
Immunodeficient individuals:
Immunosuppressive therapy Chronic lymphocytic leukemia Bone marrow transplant Multiple myeloma Acquired hypogammaglobulinemia Acquired agammaglobulinemia
Immunosuppressed patients:
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Immunoglobulin class:
ABO antibodies ISOAGGLUTININS Saline agglutinins
with optimal reactivity at 40C.
Mostly IgM.
IgG & IgA.
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Anti AB:
Group O do not have A / B antigens.
Produce anti-A, anti-B and anti-AB.
anti-AB cross-reactive antibody reacts with a
common molecular structure in both antigens.
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Anti-A1:
As per Landsteiners Law, group B and O individuals
produce anti-A.
This anti-A can be separated by absorption
procedures - anti-A and anti-A1.
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Clinical significance of ABO antibodies:
Cause both
HDFN Hemolytic disease of fetus and new born &
HTR Hemolytic transfusion reaction.
HDFN: usually presents itself with a maternal IgG
antibody to an antigen on the surface of the babys red cells.
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ABO HDFN:
Affects 1st pregnancy.
MC: O mother with A baby.
Rh HDFN:
Sensitization occurs in 1st pregnancy and affects subsequent positive pregnancies.
Rh negative mother Rh positive baby.
-
Hemolytic transfusion reaction:
Occurs when a recipient is transfused with red cells
that are an ABO group incompatible with the
antibodies in his or her serum.
Because of the complement-binding ability of the ABO
antibodies, this is always a life-threatening situation.
As the recipient antibodies react with the
incompatible red cells, complement is activated and
in vivo hemolysis, agglutination, and red blood cell
destruction occurs.
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RH BLOOD GROUP SYSTEM
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INTRODUCTION:
One of the most polymorphic and antigenic blood group
systems.
2nd only to ABO in importance in:
Blood transfusion &
A primary cause of HDFN.
The principal antigen is D and the terms Rh positive or
Rh negative refers to presence / absence of this antigen.
Other 4principal antigens are C, c, E and e.
50 other rare antigens detected.
Rh negative phenotype common in Caucasians 15 17%.
95% Indian population: Rh Positive.
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GENES:
Chromosome 1.
2 genes: RhD & RhCE.
The proteins encoded by these 2 differ by 32 to 35
AAs. That is why RhD is so antigenic in Rh negative
persons.
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NOMENCLATURE:
Discovered in the 1940s.
3 systems:
Fisher & Race: 3 closely linked genes D at one
locus, C / c at the 2nd
locus & E / e at the 3rd
locus.
Modified Weiner terminology: Supposes a single
gene.
ISBT terminology: Assigns each antigen a number
D: Rh1, C: Rh2, E:Rh3, c:Rh4 & e:Rh5.
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D ANTIGEN:
Very antigenic D+ for presence / D- for absence.
Variants: due to a point mutation causing single AA
differences.
1. Weak D [formerly Du, obsolete now]: 1 to 57
types: Type 1 to 3 90% cases.
2. Del: Not detected by routine testing but requires
adsorption-elution studies / molecular RHD
genotyping.
3. Partial D: Due to hybrid genes portion of RHD is replaced by corresponding portion of RHCE gene.
The RBCs type D+ve but make anti-D following transfusion / pregnancy.
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Rh null:
No Rh system antigens.
2 pathways:
Rh-negative person (lacking RHD) who also has an
inactive RHCE gene, referred to as an Rhnull amorph.
More often, inheritance of inactive RHAG gene,
referred to as an Rhnull regulator. RhAG protein is
required for expression and trafcking of RhCE and
RhD to the RBC membrane.
The serum of the people who form these antibodies
agglutinates cells from all people except another
Rhnull.
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Rh ANTIBODIES: Principally RBC stimulated.
Most are of the IgG class, usually the IgG1 or IgG3 subclass.
Can cross placenta.
May occur in mixtures with a minor component of IgM.
The antibodies usually appear between 6 weeks and 6
months after exposure to the Rh antigen.
IgG Rh system antibodies react best at 370C and are
enhanced when enzyme-treated RBCs are tested.
D is the most immunogenic of the common Rh antigens,
followed in decreasing order of immunogenicity by c, E, C,
and e.
30% to 85% of D-ve persons who will make anti-D following
exposure to D+ve RBCs Responders.
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LABORATORY DETERMINATION OF THE ABO SYSTEM
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-
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RBC PRECURSOR STRUCTURE
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Glucose
Galactose
N-acetylglucosamine
Galactose
Precursor
Substance
(stays the
same)
RBC
-
FORMATION OF H Ag
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Glucose
Galactose
N-acetylglucosamine
Galactose
H antigen
RBC
Fucose
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FORMATION OF A Ag
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Glucose
Galactose
N-acetylglucosamine
Galactose
RBC
Fucose N-acetylgalactosamine
-
FORMATION OF B Ag
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Glucose
Galactose
N-acetylglucosamine
Galactose
RBC
Fucose Galactose
-
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ANTISERUM
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An antiserum is a purified, diluted and standardized
solution containing known antibody, which is used
to know the presence or absence of antigen on cells
and to phenotype ones blood group.
Antiserum is named on the basis of the antibody it
contains:
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Antisera Antibody present
Anti-A anti-A
Anti-B anti-B
Anti-AB anti-A & anti-B
Anti-D anti-D
-
Sources:
MONOCLONAL ANTIBODIES
Animal inoculation.
Serum from an individual who has been sensitized to
the antigen through transfusion, pregnancy or
injection.
Serum from known blood group persons.
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Criterias:
Antiserum must meet certain criterias to be acceptable for
use.
Qualities of a good antisera:
Specific: does not cross react, and only reacts with its
own corresponding antigen,
Avid: the ability to agglutinate red cells quickly and
strongly and,
Stable: maintains it specificity and avidity till the expiry
date.
It should also be clear [as turbidity may indicate bacterial
contamination] and free of precipitate and particles.
It should be labelled and stored properly.
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Manifestation of Ag-Ab reaction:
The observable reactions resulting from the
combination of a red cell antigen with its
corresponding antibody are agglutination and/ or
haemolysis.
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Agglutination:
Is the clumping of particles with antigens on their surface,
such as erythrocytes by antibody molecules that form
bridges between the antigenic determinants.
Hemagglutination.
Agglutinogen antigen.
Agglutinin antibody.
Two stages:
1. Sensitization: Abs attach to the Ags on RBC
Sensitized RBC.
2. Agglutination: Ab binding to Ag on >1 RBC Lattice
formation.
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Hemolysis:
Ab Hemolysin.
Complement mediated lysis of the RBC membrane
with release of Hb to stain the plasma.
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Right condition for the RBCs to agglutinate / hemolysis:
1. Ab size:
Zeta potential keeps RBCs 25 nm apart.
IgG Ab max span 14 nm so can only bind to Ag
and sensitize them [can not cause agglutination] in
saline media.
IgM Ab larger and pentameric can bridge a wider
gap cause agglutination.
2. pH: Optimum pH 7.0.
3. Temperature: Optimum temperature varies
depending upon Ab type: IgG 370C, IgM 4 220C.
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Right condition for the RBCs to agglutinate / hemolysis:
4. Ionic strength:
Low ionic strength increases agglutination.
LISS 0.2% NaCl in 7% glucose is used.
5. Number of Ag sites:
Seen that IgG Abs of Rh system fail to agglutinate
RBCs suspended in saline where as IgG Abs of ABO
system can agglutinate because number of ABO
sites are 100 times more in D sites.
6. Centrifugation: at high speed attempts to overcome
the problem of distance in sensitized cells by
physically forcing the cells together.
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Right condition for the RBCs to agglutinate / hemolysis:
7. Enzyme treatment:
Treatment with weak proteolytic enzymes [Trypsin /
Papain] removes surface sialic acid residue on RBC
lowers zeta potential promotes agglutination.
Has a disadvantage destroys some blood group Ags.
8. Colloidal suspension: [Bovine albumin]
Can reduce the zeta potential helps IgG Abs of Rh
system to agglutinate.
9. Ratio of Ag & Ab:
Excess Ab Prozone phenomenon Use serial dilutions
of the antisera.
Avoid heavy RBC suspension as it may mask the presence
of a weak Ab.
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ABO GROUPING TECHNIQUES
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METHODS:
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REAGENTS:
Anti-A antibodies.
Anti-B antibodies.
Anti-AB antibodies (optional).
Group A & B RBCs.
Slides, or Test tubes.
Wooden applicator.
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IMPORTANT THINGS TO FOLLOW:
Verify that patient information on the sample
matches information on the worksheet.
Centrifuge the sample and remove the serum to a
labelled tube.
Prepare a washed 2 - 5% RBC suspension.
Use Patient cell suspension in Forward typing.
Use Patient serum for confirmation Reverse
grouping or backtyping.
At the End, Discard all materials in the isolation
trash containers.
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ABO GROUPING: RULES FOR PRACTICAL WORK:
1. Perform all tests according to the manufacturers
direction.
2. Always label tubes and slides fully and cleanly.
3. Do not perform tests at temperature higher than
room temperature.
4. REAGENT ANTISERA SHOULD BE TESTED DAILY
WITH ERYTHROCYTES OF KNOWN ANTIGENICITY.
This eliminates the need to run individual controls
each time the reagents are used.
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ABO GROUPING: RULES FOR PRACTICAL WORK:
5. Do not rely on colored dyes to identify reagent
antisera.
6. Always add serum before adding cells.
7. Observe for agglutination against a welllighted
background, and record results immediately after
observation.
8. Use an optical aid to examine reactions that appear
to the naked eye to be negative.
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ABO GROUPING: PREPARATION OF RBC SUSPENSION:
Important to the accuracy of testing in the blood
bank.
Can be prepared directly from anticoagulated blood
or from packed red cell (after separating the serum
or plasma).
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ABO GROUPING: PREPARATION OF RBC SUSPENSION:
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Procedure: (as an example preparation of 2% RBC
suspension of 10 ml volume):
Place 1 to 2ml of anticoagulated blood in a test tube.
Fill the tube with saline and centrifuge the tube.
Aspirate or decant the supernatant saline.
Repeat (steps 2 and 3) until the supernatant saline is
clear.
Pipette 10 ml of saline in to another clean test tube.
Add 0.2 ml of the packed cell button to the 10 ml of
saline.
Cover the tube until time of use. Immediately before
use, mix the suspension by inverting the tube several
times until the cells are in suspension.
-
DIRECT ABO GROUPING:
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DIRECT ABO GROUPING: PRINCIPLE:
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The direct blood grouping also called
Cell grouping / Forward grouping
employs known anti sera to identify the antigen
present or their absence on an individuals RBC.
It can be performed by the
Slide or Test tube method.
-
DIRECT ABO GROUPING: SLIDE METHOD:
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Make rings on the slide and label one ring as anti- A
and the other ring as anti-B.
First add corresponding anti- sera to the rings.
Add 10% cell suspension to both rings.
Mix using separate applicator sticks.
Observe the reaction within 2 minutes by rotating
the slide back and forth.
Interpret the results.
-
Strong agglutination of
RBCs in the presence of
any ABO grouping reagent
constitutes a positive
result.
A smooth suspension of
RBCs at the end of 2
minutes is a negative
result.
Samples that give weak or
doubtful reactions should
be retested by Tube test
ABO grouping.
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DIRECT ABO GROUPING: TEST TUBE METHOD:
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Take two tubes, label one tube anti- A and the second
anti- B.
First add corresponding anti- sera to the tubes.
Put one drop of the 2-5% cell suspension to both tubes.
Mix the antiserum and cells by gently tapping the base of
each tube with the finger or by gently shaking.
Leave the tubes at RT for 5 minutes.
Centrifuge at low speed (2200-2800 rpm) for 30 seconds.
Read the results by tapping gently the base of each tube
looking for agglutination or haemolysis against a well
lighted white background.
Interpret the results.
-
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- Prepare 2-5% cell suspension
- Label Test tubes
- Add 2 drops of Anti sera A, B , and D
-
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- Add one drop of 2-5% Patient Red Blood Cell suspension.
- Mix the contents of the tubes gently and centrifuge for 15-30 seconds at approx. 900-1000 x g
- Gently resuspend the RBCs buttons and examine for agglutination
-
TUBE METHOD READING OF RESULTS:
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Interpretation Reaction of cells
tested with
ABO Group Cell Ag Anti-B Anti-A
O No Ag - -
A A - +
B B + -
AB A, B + +
-
INDIRECT ABO GROUPING:
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INDIRECT ABO GROUPING: PRINCIPLE:
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The indirect blood grouping also called
Serum grouping / Reverse grouping
employs RBCs possessing known antigen to identify
the type of antibodies present or absent in the serum
of an individual.
It can be performed by the Test tube method.
Slide reverse grouping is not reliable.
-
INDIRECT ABO GROUPING: TEST TUBE METHOD:
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Take two tubes, label A- Cells and B cells.
Put one drop of the serum to be tested each tube.
Add one drop of 2-5% A cells to the tube labeled A cells
and one drop of 2-5% B cells to the tube labeled B cells.
Mix the contents of the tubes.
Leave the tubes at RT for 5 minutes.
Centrifuge at low speed (2200-2800 rpm) for 30 seconds.
Read the results by tapping gently the base of each tube
looking for agglutination or haemolysis against a well
lighted white background.
Interpret the results.
-
Agglutination in any tube of RBCs test or hemolysis or
agglutination in serum tests constitutes positive test
results.
A smooth suspension of RBCs after resuspension of
an RBCs button is a negative result.
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INDIRECT ABO GROUPING: INTERPRETATION:
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SERUM TESTED WITH
BLOOD GROUP
A CELL B CELL
Negative Positive A
Positive Negative B
Negative Negative AB
Positive Positive O
-
INTERPRETATION OF BOTH:
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Interpretation Reaction of serum tested
against
Reaction of cells
tested with
ABO Group O cells B Cells A cells Anti-B Anti-A
O - + + - -
A - + - - +
B - - + + -
AB - - - + +
-
OTHER METHODS OF BLOOD GROUPING:
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GEL CARDS:
Gel Cards containing Anti-A, Anti-B, and Anti-A1B are used
to test patient or donor red blood cells for the presence or
absence of the A and/or B antigens.
The results of red blood cell grouping should be confirmed
by reverse (serum) grouping, i.e. testing the individuals
serum with known A1 and B red blood cells.
In the Gel Test, the specific antibody (Anti-A, Anti-B, or
Anti-D) is incorporated into the gel. This gel has been pre-
filled into the microtubes of the plastic card. As the red
blood cells pass through the gel, they come in contact with
the antibody. Red blood cells with the specific antigen will
agglutinate when combined with the corresponding
antibody in the gel during the centrifugation step.
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GEL CARDS: INTERPRETATION OF RESULTS
A positive reaction is recorded when red cells are
retained in or above the gel column after centrifugation
A negative reaction is recorded when a distinct button of
cells sediment to the bottom of the column after
centrifugation.
A positive reaction in the Control microtube indicates a
false positive reaction, thus invalidates the tests.
Drying, discoloration, bubbles, crystals, other artefacts,
opened or damaged seals may indicate product alteration.
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PROCEDURE:
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MICROPLATE TECHNIQUE:
Microplate techniques can be used to test for antigens on
red cells and for antibodies in serum.
A microplate can be considered as a matrix of 96 short
test tubes; the principles that apply to hemagglutination in
tube tests also apply to tests in microplate.
Add reagent and patient sample( red cells/ serum)
Incubation,
Centrifugation
Red cell resuspension,
Reading of results.
Interpretation of results.
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ABO GROUPING DISCREPANCIES
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ABO GROUPING DISCREPANCIES: TECHNICAL ERRORS
Most errors are technical in nature & can be
resolve by careful repeating the test procedure.
Common errors are:
1. Contaminated reagents.
2. Dirty glass ware.
3. Over / Under centrifugation.
4. Incorrect serum:cell ratio.
5. Incorrect incubation temperature.
6. Failure to add test specimen / reagents.
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ABO GROUPING DISCREPANCIES: NON-TECHNICAL ERRORS
If after careful repeat the same agglutination
pattern is obtained than the causes can be:
1. Missing / Weak reacting Abs.
2. Missing / Weak Abs.
3. Additional Ab.
4. Plasma abnormalities.
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ABO GROUPING DISCREPANCIES: MISSING / WEAK Abs. 1. Age:
Infants before producing own Abs or who possess passively
acquired maternal Abs.
Elderly persons whose Ab levels have declined.
2. Hypogammaglobulinemia:
Lymphoma.
Leukemia.
Immunodeficiency disorders / Use of immnosuppressive drugs.
Following BM transplantation.
RESOLUTION: enhancing reaction in reverse grouping by
incubating test serum with RBCs at RT for 15 mins / at 40C or 16
0C
for 15 mins.
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ABO GROUPING DISCREPANCIES: MISSING / WEAK Ags.
1. Subgroups of A / B Ags. [RESOLUTION: Subgroup the sample.]
2. Diseases like Leukemia: ABO Ags greatly depressed.
[RESOLUTION: Investigate the diagnosis.]
3. Blood group specific substances: Ovarian cysts / carcinomas.
[RESOLUTION: Wash the cells in saline.]
4. Acquired B Ag: Effect of bacterial enzymes & adsorption of
bacterial polysaccharide on to the group A / O RBCs B
specificity weak B Ag reaction in the forward grouping.
[RESOLUTION: Acidify the anti-B reagent to pH 6 rules out
acquired B.]
5. Additives to sera. [RESOLUTION: Wash the cells in saline.]
6. Mixtures of blood: recent BT / BM transplant recipient.
[RESOLUTION: Investigate.]
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ABO GROUPING DISCREPANCIES: ADDITIONAL Ab.
1. AutoAb.: Cold autoAb - spontaneous agglutination of the A & B
cells in reverse grouping. Warm AIHA patients may have RBCs
coated with sufficient Ab to promote spontaneous
agglutination.
[RESOLUTION: Wash RBCs in warm 370C to establish cold Abs. Treat
cells with Chloroquine diphosphate to eliminate bound warm Abs]
1. Anti A1: A2 & A2B individuals may produce naturally occurring
anti-A1 which cause discrepant ABO typing.
[RESOLUTION: Investigate the diagnosis.]
1. Irregular Ab: Irregular antibodies in some other blood group
system may be present that react with antigens on the A or B
cells used in reverse grouping.
[RESOLUTION: Use A & B cells that are negative for corresponding
Ag.]
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ABO GROUPING DISCREPANCIES: PLASMA ABNORMALITIES
1. Increased globulin.
2. Abnormal proteins.
3. Whartons jelly.
All these cause increased rolueaux formation that can be
mistaken as agglutination.
[RESOLUTION: Wash with NS to remove proteins.]
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LABORATORY DETERMINATION OF THE RH SYSTEM
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Direct Slide / Tube testing method.
No Indirect / Reverse grouping.
No naturally occurring Rh antibodies are not found
in the serum of persons lacking the corresponding
Rh antigens.
In performing Rh grouping:
The number of drops,
Time &
Speed of centrifugation shall be determined by
manufacturers directions.
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Rh TYPING: SLIDE TEST METHOD
Place a drop of anti- D on a labelled slide.
Place a drop of Rh control (albumin or other control
medium) or another labelled slide.
Add two drops of 40-50% suspension of cells to each slides.
Mix the mixtures on each slide using separate applicator
sticks, spreading the mixture evenly over most of the slide.
Interpretation or results:
Agglutination of red cells- Rh positive.
No red cell agglutination- Rh negative.
A smooth suspension of cell must be observed in the
control.
Note: Check negative reactions microscopically.
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Rh TYPING: TUBE TEST METHOD
Make a 2-5% red cell suspension.
Mark D on a test tube and add two drops of anti-D.
Place a drop of Rh control (albumin or other control
medium) on another labelled tube.
Add one drop of a 2-5% cell suspension to each tube.
Mix well and centrifuge at 2200-2800 rpm for 60 seconds.
Gently resuspend the cell button and look for agglutination
and grade the results (a reaction of any grade is interpreted
as Rh positive) a smooth suspension of cells must be
observed in the control.
Collect a weakly positive and negative sample to perform
the Du test.
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Rh TYPING: Du TYPING USING IAT
Use the initial Rh D typing tube and control in procedure
above and incubate the Rh - negative or weakly reactive
samples and the control at 370C for 30 minutes.
Wash cells in both test and control tube 3-4 times with
normal saline.
Add one drop of the poly specific anti- human globulin
(Coombs) to each tube and mix well.
Centrifuge at 2200-2800 rpm for 10 second.
Gently suspend the cell button and observe for
agglutination.
Interpretation: the positive result is agglutination in the
tube containing antiD and the control is negative. A
negative result is absence of agglutination in both the test
& control.
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THE ANTI-GLOBULIN TEST
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Introduced in to clinical medicine by Coombs in 1945.
It is a sensitive technique in the detection of
Incomplete antibodies,
Antibodies that can sensitize but which fail to
agglutinate RBCs suspended in saline at room
temperature, mainly IgG.
Complements.
PRINCIPLE:
Anti-IgG / Anti-C3 in antiglobulin serum agglutinates the
incomplete Abs / Sensitizing Abs on neighboring RBCs
/ Complements by cross-linking them.
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-
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AHG Reagent:
It is made by injecting rabbits, sheep or goat with
purified human IgG or C3.
The reagent may be polyspecific or monospecific.
Polyspecific Anti-human Globulin: contains a blend
of Anti-IgG & Anti-C3b, -C3d and sometimes C4
Monospecific reagents: contains Anti-IgG alone or
Anti-C3b,-C3d alone
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Preparation of coombs control check cells (CCC):
Positive Control:
Sensitized O Rh (D) positive cells.
Negative Control:
Sensitized 0 Rh (D) negative cells.
Unsensitized 0 Rh (D) positive cells.
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Preparation of Positive Coombs control check cells (CCC):
Take 0.5 mL of 5-6 times washed and packed 0 Rh (D)
+ve red cells in a test tube.
Add 2-3 drops of IgG anti-D (select a dilution titre
[1:4] of anti-D which coats the red cells but does not
agglutinate them at 37C).
Mix and incubate at 37C for 30 minutes. If there is
agglutination, repeat the procedure using more
diluted anti-D.
Wash 3-4 times and make 5% suspension in saline for
use.
Perform a DAT which should give a 2+ reaction. If no
agglutination occurs, repeat using less diluted anti-D.
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Preparation of Negative Coombs control check cells (CCC):
0 Rh(D) negative sensitized red cells are also prepared
by treating 0 Rh(D) negative cells in the same manner.
The preparation should give a negative direct
antiglobulin test (DAT).
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TYPES:
1. Direct Antiglobulin Test [Direct Coombs Test]
DAT.
2. Indirect Antiglobulin Test [Indirect Coombs Test]
IAT.
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DAT:
It is used to demonstrate whether RBCs have been
sensitized (coated) with antibody or complement in vivo,
as in case of
HDN,
Autoimmune haemolytic anemia,
Drug induced haemolytic anemia, and
Transfusion reactions.
Principle:
Patients erythrocytes are washed to remove free plasma
proteins and directly mixed with AHG, and if incomplete
antibodies are present, agglutination occurs.
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-
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DAT:
The direct antiglobulin test (DAT) detects sensitized
red cells with IgG and/or complement components
C3b and C3d in vivo.
In vivo coating of red cells with IgG and/or
complement may occur in any immune mechanism is
attacking the patient's own RBC's.
This mechanism could be autoimmunity,
alloimmunity or a drug-induced immune mediated
mechanism.
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DAT: Requirements
Requirements:
Test tubes: (10x75 mm)
Pasteur pipettes
Incubator
Centrifuge
Reagent: Anti-human globulin serum.
Specimen: Blood drawn into EDTA is preferred but
oxalated, or clotted, citrated whole blood may be used
(specimen need not be fasting sample).
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DAT: Procedure
Prepare a 5 % suspension in isotonic saline of the red
blood cells to be tested.
With clean Pasture pipette add one drop of the prepared
cell suspension to a small tube.
Wash three times with normal saline to remove all the
traces of serum.
Decant completely after the last washing.
Add two drops of Antihuman globulin.
Mix well and incubate at 370C for 30 mins.
Centrifuge for one minute at 1500 RPM.
Resuspend the cells by gentle agitation and examine
macroscopically and microscopically for agglutination.
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IAT:
1. This test is performed to detect presence of Rh
antibodies or other antibodies in patients serum in
case of the following:
a. To check whether an Rh-negative women (married
to Rh-positive husband) has developed Anti Rh
antibodies.
b. Rh incompatible blood transfusions.
2. To detect Du Ag.
3. In cross-matching to detect Abs that might reduce
the survival of transfused cells.
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IAT: Principle
The serum containing antibodies is incubated with
erythrocytes containing antigens that adsorb the
incomplete antibodies. After washing to dilute the
excess antibody in the serum, the addition anti
globulin serum produces agglutination in the
presence of incomplete antibodies.
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IAT: Procedure
Requirements:
Test tubes: (10x75 mm)
Pasteur pipettes
Incubator
Centrifuge
Specimen: Serum (need not be fasting)
Reagents:
Antihuman globulin
IgG Anti-D serum
Coombs control cells: Make a pooled O Rho (D) positive
cells from at least three different O positive blood
samples. Wash these cells three times in normal saline
(these cells should be completely free from serum with no
free antibodies).
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IAT: Procedure
1. Label three test tubes as T (test serum) PC
(Positive control) and NC (negative control).
2. In the tube labelled as T, add two drops of Test
serum.
3. In the tube PC add two drops of 1:4 diluted IgG
Anti-D.
4. In the tube NC add two drops of NS.
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IAT: Procedure
5. Add two drops of 5 % saline suspension of the
pooled O Rh (D) positive cells in each tube.
6. Incubate all the three tubes for one hour at 37C.
7. Wash the cells three times in normal saline to
remove excess serum with no free antibodies, (in the
case of inadequate washings of the red cells,
negative results may be obtained).
8. Add two drops of Coombs serum (anti human
globulin) to each tube. Keep for 5 minutes and then
centrifuge at 1,500 RPM for one minute.
9. Resuspend the cells and examine macroscopically as
well as microscopically.
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IAT: Interpretation
Observation Conclusion
PC
Agglutination. Correct test.
No agglutination. Incorrect test, repeat.
NC
No agglutination. Correct test.
Agglutination. Incorrect test, repeat.
Test
Agglutination. Positive Patient serum contains Ab.
No agglutination. Negative.
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Factors affecting sensitivity of IAT
Temperature:
Optimal temperature: 370C.
Incubation at higher / lower temperature may give false
positive results.
Serum Cell ratio: Increasing the ratio of serum to cells
increases the antibody coating. Commonly used ratio in
saline suspension is 2:1 but in LISS suspending cells, use
equal volume of serum and 2% cell suspension.
Incubation time:
Saline, Albumin or enzyme technique : 45-60 minutes.
LISS suspended cells - Routine 15 minutes.
Suspension medium: The sensitivity of IAT can be
increased with addition of 22% bovine albumin, enzyme or
by using LISS suspended cells.
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False Negative Antiglobulin test results:
1. Inadequate cell washing.
2. Delay in adding antiglobulin reagent after the
washing step.
3. Presence of small fibrin clots among the cells.
4. Inactive, or forgotten antiglobulin reagent .
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False Positive Antiglobulin test results:
1. Using improper sample (clotted cells instead of
EDTA for Direct Antiglobulin Test, DAT).
2. Spontaneous agglutination (cells heavily coated with
IgM).
3. Non-specific agglutination ("sticky cells").
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CROSS-MATCHING
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Purpose:
1. To select donors blood that will not cause any
adverse reaction like hemolysis or agglutination in
the recipient.
2. Also to help the patient to receive maximum benefit
from transfusion of red cells, which will survive
maximum in his circulation.
However, a cross match will not prevent
immunization of the patient, and will not guarantee
normal survival of transfused erythrocytes or detect
all unexpected antibodies in a patients serum.
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Types of Cross Match:
1. Major.
2. Minor.
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Major Cross Match:
RECIPIENTS / PATIENTS SERUM with DONORs RBCs.
It is much more critical for assuring safe transfusion
than the minor compatibility test.
It is called major because the antibody with the
recipients serum is most likely to destroy the donors
red cells and that is why it is called major cross
match.
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Minor Cross Match:
DONORs SERUM with PATIENTs RBCs.
It is usually thought that any antibody in the donors
serum will be diluted by the large volume of the
recipients blood, so it causes relatively less problem
and so called minor cross match.
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Selection of blood for Cross Match:
When whole blood is to be transfused, the blood
selected for cross- match should be of the same
ABO and Rh (D) group as that of the recipient.
However, Rh positive recipients may receive
either Rh positive or Rh negative blood.
Group A is the second choice of blood because anti-B in Gp
A blood is likely to be weaker than anti-A in Gp B blood.
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Group of
Patient
Choice of Blood
1st 2
nd 3
rd 4
th
A A O --- ---
B B O --- ---
O O --- --- ---
AB AB A* B O
-
Procedure of Cross Match:
4 PHASES:
1. Saline.
2. Protein.
3. AHG.
4. Enzyme.
1. Saline tube technique at RT: provides the optimum
temperature and medium for the detection of IgM
antibodies of ABO system and other potent cold
agglutinins.
2. Saline 370C: is the optimum for the detection of warm
agglutinin, of which are saline reactive IgG antibodies of
the Rh/ Hr system.
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Procedure of Cross Match:
3. AHG: is highly efficient for the detection of most kinds of
incomplete antibodies.
4. Enzyme technique- is a very sensitive one for the
detection of some low affinity Rh antibodies, which are
not detected by other methods including the antiglobulin
technique.
Procedure:
1. Put 3 drops of patients serum in to a test tube.
2. Put one drop of donors 3% red cells suspension.
3. Mix and centrifuge at 3400 rpm for 15 seconds.
4. Examine for agglutination or haemolysis, if compatible
proceed with the next phase.
5. Mix the contents of the tube and incubate at 370C for 20
30 min.
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Procedure of Cross Match:
Note: potentiators such as a drop of 22% albumin may be
added at this phase to increase the sensitivity of the test.
6. Centrifuge at 3400 rpm for 15 seconds and examine for
agglutination or hemolysis. If there is no hemolysis or
agglutination proceed with the next phase.
7. Wash the contents of the tube 3-4 times with normal
saline.
8. After the last wash, decant all saline and add two drops of
AHG reagent and mix.
9. Centrifuge at 3400 rpm for 15 seconds.
10. Gently re suspend the cells button and examine
macroscopically and microscopically for agglutination or
hemolysis.
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Procedure of Cross Match:
Enzyme cross match can be performed by using different
enzymes:
Bromelin / Ficin / Papain / Trypsin.
Two methods are available to carry out enzyme cross
match:
One stage &
Two stage methods.
The one-stage technique involves enzyme, patients serum
and donors red cell incubated together.
The two-stage technique involves red cells pre-treated with
enzyme and then tested with the patients serum.
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